Trialuminium bismuth hexaoxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Details on species / strain selection:

Standard species as per OECD guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately eleven weeks old and females approximately 14 weeks old.
- Weight at study initiation: Males weighed 290 to 356 g and females weighed 206 to 241 g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (Teklad Global Certified Diet).
- Water (e.g. ad libitum): Mains drinking water freely available.
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for food available showing acceptable for use and water routinely analysed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): At least 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Details on route of administration:

Standard as per OECD guideline and most relevant for test substance.

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Distilled water. Due to the nature of the analytical method, stability of the test item formulations was not determined. The test item formulations were prepared daily and dosed within two hours of formulation; formulations are assumed to have been stable for this duration. The homogeneity of the test item formulations were determined.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique. The test item formulations were weighed into glass beakers and then dried in an oven at approximately 105°C before allowing to cool over silica gel in a desiccator and the residue weighed. Accuracy and precision samples were prepared and the method was validated by analysing five recovery samples at nominal 1 and 200 mg/mL. Homogeniety was also assessed. The concentrations in the dose formulations were analysed on 2 occassions during the study using the same method.

Duration of treatment / exposure:

Males were dosed daily prior to mating for two weeks which continued until their scheduled necropsy on day 44 or 45.
Females were dosed daily prior to mating for two weeks which continued until their scheduled necropsy on day 14 post partum.

Frequency of treatment:

Daily by oral gavage

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

12 males and 12 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a dose range finder.
- Rationale for animal assignment (if not random): Animals randomly assigned

Positive control:

Not a guideline requirement

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION/EFFICIENCY:
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from five males/females from each dose group for haematological assessments on Day 43 and postpartum day 13 respectively (where possible).
- Anaesthetic used for blood collection: No (samples taken from lateral tail vein).
- Animals fasted: No
- How many animals: 5 males and 5 females per dose group.
- Parameters: Haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration), total leukocyte count, differential leukocyte count (neutrophils, lymphocytes, monocytes, easinophils and basophils), platelet count, reticulocyte count prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from five males/females from each dose group for blood chemical assessments on Day 43 and postpartum day 13 respectively (where possible).
- Animals fasted: No
- How many animals: 5 males and 5 females per dose group
- Parameters: Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: All dose groups
- Battery of functions tested: behavioural assessment (gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation). Motor activity, forelimb/hindlimb grip strength and sensory reactivity (grasp response, vocalization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex and startle reflex).

IMMUNOLOGY: Yes (thyroid hormone analysis)
- Time schedule for examinations: At scheduled termination
- How many animals: Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
- Dose groups that were examined: All
- Parameters: Thyroxine (T4)

ORGAN WEIGHTS: Yes
- Organs weighed: From 5 males and females per dose group: adrenals, brain, Epididymides, heart, kidneys, liver, ovaries, pituitary (weighed following partial fixation), prostate, seminal vesicles (with coagulating gland), spleen, testes, thymus, thyroid (weighed following partial fixation with parathyroid) and uterus (with cervix). From remaining animals: Epididymides, ovaries, pituitary (weighed following partial fixation), prostate, seminal vesicles (with coagulating gland), testes, thyroid (weighed following partial fixation with parathyroid) and uterus (with cervix)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

HISTOPATHOLOGY: Yes
The following tissues were processed, cut and stained with haemotoxylin and eosin from 5 males and females from the controls and the high dose group:
Adrenals, aorta, bone and bone marrow (femur), bone and bone marrow (sternum), brain, cecum, cervix, coagulating gland, colon, duodenum, esophagus, epididmymis, eyes, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph node mandibular, lymph node mesenteric, mammary gland, ovaries, pancreas, parathyroid glands, peyers patch, pituitary gland, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina and gross lesions.

The following was also prepared from all non-pregnant pairings as appropriate: cervix, coagulating glands, epididymis, ovaries, mammary gland, pituitary, prostate, seminal vesicles, testes, uterus and vagina.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring
Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with
appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Clinical signs were limited to one male and one female treated with 300 mg/kg bw/day showing red/brown staining around the snout on single occasions, and one female treated with 1000 mg/kg bw/day showing noisy respiration on a single occasion. At these low incidences these clinical signs are considered not to be toxicologically significant, and are considered incidental.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on bodyweight or body weight gains for males at 100, 300 or 1000 mg/kg bw/day throughout the study. There was no effect of treatment on bodyweight or body weight gains for females during prepairing, gestation or lactation at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption for males at 100, 300 or 1000 mg/kg bw/day throughout the study. There was no effect of treatment on food consumption for females during pre-pairing, gestation or lactation at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the gestation and lactation data did not reveal any significant intergroup differences.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption for males at 100, 300 or 1000 mg/kg bw/day throughout the study. There was no effect of treatment on food consumption for females during pre-pairing, gestation or lactation at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the gestation and lactation data did not reveal any significant intergroup differences.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Males treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in neutrophil count compared to controls. However, as only one individual value for these males exceeded the historical control range for neutrophils, and a similar effect was not observed at the higher dosages, the intergroup difference was considered not to be of toxicological significance.

All treated females showed statistically significant increases (p<0.01 – p<0.05) in hemoglobin, erythrocyte count and hematocrit, however, a true dose related response was not observed for any of these parameters. Individual values were within the historical control range for erythrocytes for all treated females, and for both hemoglobin and hematocrit, only one female treated with 100 mg/kg bw/day exceeded the historical control range. Females treated at 1000 mg/kg bw/day also showed statistically significantly lower (p<0.05) mean corpuscular hemoglobin concentration, but all individual values for these females were within the historical control normal range. These intergroup differences are therefore considered not to be of toxicological significance.

All treated males, and females treated with 300 and 1000 mg/kg bw/day showed statistically significantly higher (p<0.01) reticulocytes when compared to controls. Although the majority of these individual values exceeded the historical control range, in the absence of any histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Males treated with 300 and 1000 mg/kg bw/day showed statistically significantly lower (p<0.05) alanine aminotransferase compared to controls. However, all individual values were within the historical control range, and no associated histopathological correlates were evident. Therefore, the intergroup differences were considered to be due to normal biological variation and not to be toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

One male treated at 1000 mg/kg bw/day showed hunched posture on one occasion. In the absence of similar effects observed during the daily clinical observations or in any other animals from this treatment group, this was considered incidental and unrelated to treatment.

Immunological findings:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 1000 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of organ weights did not indicate any adverse effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Thyroid/parathyroid weights, both absolute and relative to terminal body weight, were statistically significantly lower (p<0.05) for all treated females compared to controls. In the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance. All statistically significant values were also within the ranges of historical control data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All males treated with 1000 mg/kg bw/day had colored/ black colored contents in the cecum. Eleven of these males also had black colored contents in the colon, six males had black colored contents in the rectum and one also had black colored contents in the stomach. Three females treated with 1000 mg/kg bw/day, three males treated with 300 mg/kg bw/day and two males treated with 100 mg/kg bw/day had black colored contents in the cecum. Two of the 300 mg/kg bw/day males also had black colored contents in the colon and one also had black colored contents in the rectum.

Histopathological examination of the gastro-intestinal tract did not reveal any significant microscopic changes which resulted from the black contents observed at necropsy. Therefore, these observations were considered to be nonadverse.

The following macroscopic findings observed were not associated with any treatment related microscopic abnormalities or true dose related responses and were considered to be incidental and unrelated to treatment.
- Small left testis and epididymis in one male treated with 300 mg/kg bw/day
- Sloughing of the glandular region of the stomach in one female treated with 300 mg/kg bw/day
- An enlarged spleen in one control female

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Functional performance tests did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Females from all treatment groups showed a statistically significant reduction (p<0.05) in the final 20% activity. A true dose related response was not evident and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.

Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

The following treatment-related microscopic abnormality was detected:

Stomach: Minimal vacuolation of the squamous epithelium at the limiting ridge was observed in three males treated with 1000 mg/kg bw/day. However, this was minimal and indicative of minor irritation at the point of contact. As no further changes were apparent this was considered to be non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The oral administration of the test substance to rats of both sexes by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any adverse treatment related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trialuminium bismuth hexaoxide

EC Number:

235-552-3

EC Name:

Trialuminium bismuth hexaoxide

Cas Number:

12284-76-3

Molecular formula:

Al3BiO6

IUPAC Name:

bismuth;oxido(oxo)alumane

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Wistar Han™:RccHan™:WIST

Details on species / strain selection:

Standard species as per OECD guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately eleven weeks old and females approximately 14 weeks old.
- Weight at study initiation: Males weighed 290 to 356 g and females weighed 206 to 241 g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (Teklad Global Certified Diet).
- Water (e.g. ad libitum): Mains drinking water freely available.
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for food available showing acceptable for use and water routinely analysed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): At least 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Distilled water. Due to the nature of the analytical method, stability of the test item formulations was not determined. The test item formulations were prepared daily and dosed within two hours of formulation; formulations are assumed to have been stable for this duration. The homogeneity of the test item formulations were determined.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique. The test item formulations were weighed into glass beakers and then dried in an oven at approximately 105°C before allowing to cool over silica gel in a desiccator and the residue weighed. Accuracy and precision samples were prepared and the method was validated by analysing five recovery samples at nominal 1 and 200 mg/mL. Homogeniety was also assessed. The concentrations in the dose formulations were analysed on 2 occassions during the study using the same method.

Duration of treatment / exposure:

Males were dosed daily prior to mating for two weeks which continued until their scheduled necropsy on day 44 or 45.

Females were dosed daily prior to mating for two weeks which continued until their scheduled necropsy on day 14 post partum.

Frequency of treatment:

Daily by oral gavage

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

12 males and 12 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a dose range finder.
- Rationale for animal assignment (if not random): Animals randomly assigned

Positive control:

Not a guideline requirement

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION/EFFICIENCY:
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood samples were taken from five males/females from each dose group for haematological assessments on Day 43 and postpartum day 13 respectively (where possible).
- Anaesthetic used for blood collection: No (samples taken from lateral tail vein).
- Animals fasted: No
- How many animals: 5 males and 5 females per dose group.
- Parameters: Haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration), total leukocyte count, differential leukocyte count (neutrophils, lymphocytes, monocytes, easinophils and basophils), platelet count, reticulocyte count prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood samples were taken from five males/females from each dose group for blood chemical assessments on Day 43 and postpartum day 13 respectively (where possible).
- Animals fasted: No
- How many animals: 5 males and 5 females per dose group
- Parameters:
Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: All dose groups
- Battery of functions tested: behavioural assessment (gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation). Motor activity, forelimb/hindlimb grip strength and sensory reactivity (grasp response, vocalization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex and startle reflex).

IMMUNOLOGY: Yes (thyroid hormone analysis)
- Time schedule for examinations:
At scheduled termination
- How many animals:
Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
- Dose groups that were examined: All
- Parameters: Thyroxine (T4)

ORGAN WEIGHTS: Yes
- Organs weighed: From 5 males and females per dose group: adrenals, brain, Epididymides, heart, kidneys, liver, ovaries, pituitary (weighed following partial fixation), prostate, seminal vesicles (with coagulating gland), spleen, testes, thymus, thyroid (weighed following partial fixation with parathyroid) and uterus (with cervix). From remaining animals: Epididymides, ovaries, pituitary (weighed following partial fixation), prostate, seminal vesicles (with coagulating gland), testes, thyroid (weighed following partial fixation with parathyroid) and uterus (with cervix)

Oestrous cyclicity (parental animals):

Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
Wistar rats generally have smaller litter sizes compared with other strains and accordingly no additional culling of pups was performed except those required for the Day 4 T4 analysis.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number of offspring born, number of offspring alive recorded daily and reported on days 1, 4, 7 and 13 post partum, sex of offspring from birth to Day 13 post partum, clinical condition of offspring from birth to Day 13 post partum, individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights calulated from this data).

GROSS EXAMINATION OF DEAD PUPS:
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Day 13 offspring (male/female) serum samples analysed for Thyroxine (T4).

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

HISTOPATHOLOGY: Yes
The following tissues were processed, cut and stained with haemotoxylin and eosin from 5 males and females from the controls and the high dose group:
Adrenals, aorta, bone and bone marrow (femur), bone and bone marrow (sternum), brain, cecum, cervix, coagulating gland, colon, duodenum, esophagus, epididmymis, eyes, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph node mandibular, lymph node mesenteric, mammary gland, ovaries, pancreas, parathyroid glands, peyers patch, pituitary gland, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina and gross lesions.

The following was also prepared from all non-pregnant pairings as appropriate: cervix, coagulating glands, epididymis, ovaries, mammary gland, pituitary, prostate, seminal vesicles, testes, uterus and vagina.

Postmortem examinations (offspring):

Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring
Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with
appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Reproductive indices:

Mating performance and fertility: Pre-coital interval, mating index and pregnancy index.
Gestation and parturition: gestation length and parturition index.

Offspring viability indices:

Litter responses: post implantation loss, live birth index, viability index (day 4 and 13) and sex ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Clinical signs were limited to one male and one female treated with 300 mg/kg bw/day showing red/brown staining around the snout on single occasions, and one female treated with 1000 mg/kg bw/day showing noisy respiration on a single occasion. At these low incidences these clinical signs are considered not to be toxicologically significant, and are considered incidental.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on bodyweight or body weight gains for males at 100, 300 or 1000 mg/kg bw/day throughout the study. There was no effect of treatment on bodyweight or body weight gains for females during prepairing, gestation or lactation at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption for males at 100, 300 or 1000 mg/kg bw/day throughout the study. There was no effect of treatment on food consumption for females during prepairing, gestation or lactation at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the gestation and lactation data did not reveal any significant intergroup differences.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption for males at 100, 300 or 1000 mg/kg bw/day throughout the study. There was no effect of treatment on food consumption for females during prepairing, gestation or lactation at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the gestation and lactation data did not reveal any significant intergroup differences.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Males treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in neutrophil count compared to controls. However, as only one individual value for these males exceeded the historical control range for neutrophils, and a similar effect was not observed at the higher dosages, the intergroup difference was considered not to be of toxicological significance.

All treated females showed statistically significant increases (p<0.01 – p<0.05) in hemoglobin, erythrocyte count and hematocrit, however, a true dose related response was not observed for any of these parameters. Individual values were within the historical control range for erythrocytes for all treated females, and for both hemoglobin and hematocrit, only one female treated with 100 mg/kg bw/day exceeded the historical control range. Females treated at 1000 mg/kg bw/day also showed statistically significantly lower (p<0.05) mean corpuscular hemoglobin concentration, but all individual values for these females were within the historical control normal range. These intergroup differences are therefore considered not to be of toxicological significance.

All treated males, and females treated with 300 and 1000 mg/kg bw/day showed statistically significantly higher (p<0.01) reticulocytes when compared to controls. Although the majority of these individual values exceeded the historical control range, in the absence of any histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Males treated with 300 and 1000 mg/kg bw/day showed statistically significantly lower (p<0.05) alanine aminotransferase compared to controls. However, all individual values were within the historical control range, and no associated histopathological correlates were evident. Therefore, the intergroup differences were considered to be due to normal biological variation and not to be toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

One male treated at 1000 mg/kg bw/day showed hunched posture on one occasion. In the absence of similar effects observed during the daily clinical observations or in any other animals from this treatment group, this was considered incidental and unrelated to treatment.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Functional performance tests did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Females from all treatment groups showed a statistically significant reduction (p<0.05) in the final 20% activity. A true dose related response was not evident and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance. Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

The following treatment-related microscopic abnormality was detected:
Stomach: Minimal vacuolation of the squamous epithelium at the limiting ridge was observed in three males treated with 1000 mg/kg bw/day. However, this was minimal and indicative of minor irritation at the point of contact. As no further changes were apparent this was considered to be non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day, with all females showing regular cycling. Assessment of estrous cycles at termination did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages ofthe sperm cycle).

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, as assessed by the number of paired animals that mated, was unaffected by treatment at dosages of 100, 300 or 1000 mg/kg bw/day.
With the exception of one control female all remaining animals mated within the first four days after pairing.

There was no effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 100, 300 or 1000 mg/kg bw/day. One control female, one female treated with 300 mg/kg bw/day and one female treated with 1000 mg/kg bw/day were found to be non-pregnant following positive evidence of mating. Histopathological examinations of the females and their respective male partners did not reveal any significant microscopic changes which could account for the lack of pregnancy, therefore, these were considered incidental and unrelated to treatment.

The intergroup distribution of gestation length observed during the study did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day. Group mean gestation length for females treated with 1000 mg/kg bw/day was statistically significantly longer (p<0.05) when compared to controls. However, as all individual female gestation lengths were within historical control ranges, this was considered to be due to normal biological variation, and not toxicologically significant.

In total 11, 12, 11 and 10 females from control, 100, 300 and 1000 mg/kg bw/day dose groups, respectively, gave birth to a live litter and successfully reared young to Day 13 of age. One female treated at 1000 mg/kg bw/day experienced a total litter loss post partum.

There was no effect of maternal treatment on the number of implantations, post-implantation loss and live birth index, and subsequent offspring survival to Day 13 of age at dosages of 100, 300 or 1000 mg/kg bw/day. Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.

Statistical analysis of the data did not reveal any significant intergroup differences.

Details on results (P0)

There were no treatment related or adverse effects to parental males and females under the conditions of the test.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain. Male and female offspring body weights were statistically significantly increased (p<0.05) on Day 1 in litters from females treated with 1000 mg/kg bw/day. An increase in body weight is considered not to represent an adverse effect of treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item indicated by offspring visible nipple count in male offspring on Day 13 post partum, or anogenital distance at 100, 300 or 1000 mg/kg bw/day.

Male offspring from females treated with 100 and 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in ano-genital distance. A similar effect was not evident in normalized ano-genital distance for males or in females. With the exception of one individual value from each group, the remaining individual values were within historical control ranges and in the absence of a true dose related response, the intergroup differences were considered not to be of toxicological significance.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 1000 mg/kg bw/day.

Details on results (F1)

There were no treatement related and adverse effects to F1 offspring as a result of treatment with the substance.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Tabular summary report of effects on reproduction/development

Observations		Dose level (mg/kg bw/day)
		0 (control)	100	300	1000
Paired animals	n	12M 12F	12M 12F	12M 12F	12M 12F
Females showing evidence of copulation	n	12	12	12	12
Pregnant females	n	11	12	11	11#
Conception Days 1-4	n	11	12	11	11
Conception Day 22	n	1	0	0	0
Gestation = 22 days	n	2	2	2	2
Gestation = 22.5 days	n	5	2	5	1
Gestation = 23 days	n	4	3	3	6
Gestation = 23.5 days	n	0	5	1	4
Dams with live young born	n	11	12	11	11
Dams with live young at Day 13 post-partum	n	11	12	11	10
Implants/dam	mean	13.9	12.3	13.6	12.0
Live offspring/dam at Day 1 post-partum	mean	12.5	11.3	12.6	11.1
Live offspring/dam at Day 4 BC post-partum	mean	12.4	11.1	12.6	11.0
Live offspring/dam at Day 4 AC post-partum	mean	10.4	9.5	10.6	9.5
Live offspring/dam at Day 7 post-partum	mean	10.4	9.5	10.5	9.5
Live offspring/dam at Day 13 post-partum	mean	10.4	9.5	10.5	9.5
Sex ratio: % males at Day 1 post-partum	mean	49.6	41.1	53.4	49.4
Sex ratio: % males at Day 4 BC post-partum	mean	49.2	41.3	53.4	49.2
Sex ratio: % males at Day 4 AC post-partum	mean	56.7	48.2	60.4	54.5
Sex ratio: % males at Day 7 post-partum	mean	56.7	48.2	60.7	54.5
Sex ratio: % males at Day 13 post-partum	mean	56.7	48.2	60.7	54.5
Litter weight (g) at Day 1 post-partum	mean	69.48	67.82	72.33	68.93
Litter weight (g) at Day 4 BC post-partum	mean	102.49	95.72	103.28	99.44
Litter weight (g) at Day 4 AC post-partum	mean	85.82	82.82	86.83	86.34
Litter weight (g) at Day 7 post-partum	mean	137.25	130.76	137.73	138.34
Litter weight (g) at Day 13 post-partum	mean	270.63	256.35	269.88	272.38
Male offspring weight (g) at Day 1 post-partum	mean	5.74	6.26	5.83	6.53
Male offspring weight (g) at Day 4 BC post-partum	mean	8.52	8.97	8.29	9.56
Male offspring weight (g) at Day 4 AC post-partum	mean	8.51	8.97	8.28	9.56
Male offspring weight (g) at Day 7 post-partum	mean	13.71	14.18	13.20	15.18
Male offspring weight (g) at Day 13 post-partum	mean	26.97	27.78	25.85	29.42
Female offspring weight (g) at Day 1 post-partum	mean	5.52	6.02	5.62	6.20
Female offspring weight (g) at Day 4 BC post-partum	mean	8.20	8.73	8.08	9.17
Female offspring weight (g) at Day 4 AC post-partum	mean	8.15	8.76	8.05	9.16
Female offspring weight (g) at Day 7 post-partum	mean	13.11	13.76	13.06	14.66
Female offspring weight (g) at Day 13 post-partum	mean	26.05	27.11	25.66	28.99
Loss of offspring/dam
Pre-natal (implantations minus live births)
0	n	1	7	3	4
1	n	8	1	6	4
2	n	1	3	1	2#
3	n	1	0	1	1
4	n	1	0	0	0
5	n	0	1	0	0
Post-natal (live births minus offspring alive on Day 13 post-partum) – excluding offspring culled on Day 4 post-partum.
0	n	10	10	10	9
1	n	1	2	1	2#

# includes one female with total litter loss post partum

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance to rats of both sexes by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any adverse treatment related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.